title: Combined optical and topographic imaging reveals different arrangements of human RAD54 with presynaptic and postsynaptic RAD51-DNA filaments.
abstract: Essential genome transactions, such as homologous recombination, are achieved by concerted and dynamic interactions of multiple protein components with DNA. Which proteins do what and how, will be reflected in their relative arrangements. However, obtaining high-resolution structural information on the variable arrangements of these complex assemblies is a challenge. Here we demonstrate the versatility of a combined total internal reflection fluorescence and scanning force microscope (TIRF-SFM) to pinpoint fluorescently labeled human homologous recombination protein RAD54 interacting with presynaptic (ssDNA) and postsynaptic (dsDNA) human recombinase RAD51 nucleoprotein filaments. Labeled proteins were localized by superresolution imaging on complex structures in the SFM image with high spatial accuracy. We observed some RAD54 at RAD51 filament ends, as expected. More commonly, RAD54 interspersed along RAD51-DNA filaments. RAD54 promotes RAD51-mediated DNA strand exchange and has been described to both stabilize and destabilize RAD51-DNA filaments. The different architectural arrangements we observe for RAD54 with RAD51-DNA filaments may reflect the diverse roles of this protein in homologous recombination.
authors.affiliation: Department of Genetics, Erasmus Medical Center, Rotterdam 3000 CA, The Netherlands.
authors.forename: Humberto
authors.initials: H
authors.lastname: Sanchez
authors.forename: Aryandi
authors.initials: A
authors.lastname: Kertokalio
authors.forename: Sari
authors.initials: S
authors.lastname: van Rossum-Fikkert
authors.forename: Roland
authors.initials: R
authors.lastname: Kanaar
authors.forename: Claire
authors.initials: C
authors.lastname: Wyman
mesh_terms: D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004265:DNA Helicases; D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D008853:Microscopy / Q000379:methods*; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism*; D051135:Rad51 Recombinase / Q000737:chemistry / Q000378:metabolism*; D013569:Synapses / Q000378:metabolism*
chemical_list: D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D009687:Nuclear Proteins; D004247:DNA; C495153:RAD51 protein, human; D051135:Rad51 Recombinase; D004265:DNA Helicases; C103532:RAD54L protein, human
keywords: DNA break repair; DNA–protein interaction; genome stability; image registration; single-molecule microscopy